Production fo eukaryotic proteins and nucleic acid molecules in c. elegans

ABSTRACT

Plasmid vectors for expression in  Caenorhabditis elegans  harbouring a heat inducible promoter nucleotide sequence, a synthetic intron nucleotide sequence optionally containing a Shine-Dalgarno sequence for efficient shuttling between  C. elegans  and  E. coli , optionally a nucleotide sequence coding for a nuclear localisation signal or secretion signal, a nucleotide sequence coding for a recognisable tag, optionally a nucleotide sequence coding for a fluorescent protein, a nucleotide sequence coding for a protease cleavage site, a multiple cloning site containing a nucleotide sequence coding for an eukaryotic, such as human, protein or a nucleic acid molecule and a nucleotide sequence coding for termination of translation, are described. Methods of particularly large scale production of eukaryotic, such as human, proteins and nucleic acid molecules in nematodes are also described. The methods comprise injection one or several different plasmid vectors into the gonad of  C. elegans  hermaphrodites, cultivating the nematodes in growth medium, optionally comprising labeled bacteria as feed for the nematodes, at temperatures below 25 DEG C., followed by shifting to 30-33 DEG C. for induction of eukaryotic protein or nucleic acid molecule expression in several hundred somatic cells, with highest expression levels in neuronal and epidermal cells, and isolating the eukaryotic protein or nucleic acid molecule from said cells

The present invention relates to the production of post-translationallymodified eukaryotic proteins in general and human proteins inparticular, in the nematode Caenorhabditis elegans. It also includes theproduction of post-translationally modified nucleic acid molecules, suchas tRNA. It further includes the co-translational labeling of theexpressed proteins with identifiable labels such as ²H, ¹³C, ¹⁵N,Se-methionine, Se-cystein or non-natural amino acids. Similarly thelabeling of nucleic acid molecules with ²H, ¹³C and ¹⁵N.

BACKGROUND

There are several alternatives for the production of eukaryotic proteinsin different expression systems. The following expression systems arecurrently in use.

Bacteria

Many E. coli expression systems are commercially available. Someexamples are pET (Promega), pOE (Qiagen), pGEX (Amersham Pharmacia),ptrcHIS (Invitrogen), pDUAL (Stratagene). The advantage of E. colisystems are that they are cheap and very easy to use. The maindisadvantage is that many eukaryotic proteins do not fold properly whenexpressed in E. coli and form insoluble aggregates. Codon usage is verydifferent from that in higher eukaryotes. Often eukaryotic proteins mustbe modified following translation in order to be able to fold into theproper structure and/or to become activated. E. coli is not able tocarry out complex post translational modifications such as acetylation,N- and O-linked glycosylation and, acylation and phosphorylation whichare exclusively performed by eukaryotic cells.

The levels of expression vary enormously from protein to protein. Theyield of recombinant protein from 1 liter of E. coli culture amountstypically to about 10 mg. In rare cases amounts of hundreds ofmilligrams of recombinant protein per liter E. coli culture can beobtained.

Yeast

The yeast Pichia pastoris is a well established system for expressingrecombinant proteins. Several companies sell the relevant plasmidvectors. Invitrogen Corp. for example sells the pPIC set of plasmids.The advantages of Pichia is that being a eukaryote, thepost-translational modifications are more similar to those that occur inhumans or higher eukaryotes. Pichia is easy and fast to transform. It isalso easy to grow on a large scale. Expression levels vary considerably.Levels as high as 12 grams/liter have been reported.

Insect Cells

Insect cells can also be used to express recombinant proteins. Severalcompanies sell the relevant plasmid vectors and cell lines. Invitrogen'ssystems are DES, InsectSelect and MaxBac. The advantages of insect cellsare that, being multicellular eukayotes, insects are much more likehumans than yeast are. The disadvantages are (i) insect cells are muchmore difficult to grow and maintain than bacterial or yeast cells (ii)they are more expensive to cultivate (iii) they require sterileincubators (iv) the expression levels are much lower than those seenwith the yeast or bacterial systems.

Human Cell Lines

Human cell lines can also be used, but they require growth factors inorder to keep them alive. Today, human growth factors are veryexpensive, and this makes human cell lines bad candidates for e.g.growth factor production.

Nematodes

Nematodes, small roundworms, are one branch of eukaryotic organisms thathave so far not been exploited for large-scale protein or nucleic acidproduction. Nematodes are very simple animals and have served as adevelopmental model system ever since 1949 (Dougherty E C and Nigon V.,J. Parasitol. (1949) 35, 11; Brenner S in a letter to Max Perutz, 5 Jun.1963). Especially the development of each of the 959 cells in thenematode C. elegans is well characterized and its entire genome wasrecently sequenced and is now publicly available (“The C. elegansSequencing Consortium” Genome Sequence of the nematode C. elegans: Aplatform for investigating biology. Science (1998) 282, 2012-2018).Nematodes are eukaryotic organisms that are genetically much moreclosely related to humans than bacteria, about 60% of their proteins arehomologous to human proteins.

What makes nematodes interesting as a protein expression system forproduction of eukaryotic and in particular human proteins is the factthat they are equipped with the necessary machinery to performpost-translational modifications on proteins. Hence, proteins (peptidedrugs) produced in nematodes are virtually identical to the naturalhuman proteins and as a result may have fewer undesirable side effectsand a higher specific activity requiring lower dosages. Other advantagesof the nematode expression system include high yields of expressedprotein, its low maintenance costs, its ease of use and that one caneasily scale up the production.

Expression of human beta-amyloid peptide in transgenic C. elegans toproduce muscle-specific deposits immunoreactive with anti-beta-amyloidpolyclonal and monoclonal antibodies has been described by C. D. Link(Link C. D., Expression of human beta-amyloid peptide in transgenicCaenorhabditis elegans, Proc. Natl. Acad. Sci.(1 995) 92,9368-9372), andhe suggests that his invertebrate model may be useful for in vivoinvestigation of factors that modulate amyloid formation.

The international patent application WO 00/54815 discloses expression ofDNA or proteins in C. elegans by using an expression vector comprising apromoter that directs the gene expression to the excretory cell of C.elegans. The reason for the protein expression is not production andisolation of a protein but for discovery of novel molecules, i.e. drugs,involved in the cell motility, cell shape and cell outgrowth process,and to establish their function.

DESCRIPTION OF THE INVENTION

The present invention provides an in vivo expression system forproduction of eukaryotic, such as human, proteins and nucleic acidmolecules that is easy to handle, inexpensive, genetically stable andeasy to scale up. The nematode expression system of the invention issuitable for the large-scale production of ultra pure recombinant humanproteins. Proteins and nucleic acid molecules produced will contain allthe modifications that are typical for higher organisms (eukaryotes),such as acetylation, N- and O-linked glycosylation and, acylation,phosphorylation and cleavage of signal sequences etc. Thesemodifications are crucial for the specificity of a medically interestingprotein in signaling pathways.

The novel nematode protein expression system combines the advantages ofeukaryotic cells such as post-translational modifications with thesimplicity of handling known from E. coli fermentation.

The expression system comprises the nematode Caenorhabditis elegans.Since the number of different plasmids that can be simultaneouslyinjected into C. elegans is in excess of twenty, this enables thesimultaneous expression of multiple plasmids harboring e.g. differentsubunits of large complexes containing proteins and/or nucleic acids.

Examples of eukaryotic proteins that may be produced on an industrialscale with the present invention include: human growth factors, growthfactor receptors (membrane bound or soluble part) for basic research onstem cells and for medical applications such as stem cell basedtreatment of heart disease, diabetes, cancer, and diseases of thenervous system, including Parkinson's and Alzheimer's disease. Inaddition monoclonal antibodies, G-proteins, G-protein coupled receptors,and large, multi-subunit protein-RNA complexes such as polymerases,telomerase and splicing factor complexes. Beside potential medicalapplications, the produced proteins and nucleic acids can be used forstructure characterization by X-ray crystallography, electroncrystallography or NMR where one studies post-translational modifiedproteins and nucleic acids. The C. elegans expression system can also beused for labeling of eukaryotic proteins with ²H, ¹³C, ¹⁵N, Se-Met,Se-Cys or non-natural amino acids for crystallographic applications orwith ²H, ¹³C, or ¹⁵N, for NMR experiments.

One aspect of the present invention is directed to a plasmid vector forexpression in Caenorhabditis elegans comprising in the 5′ to 3′direction of transcription operably linked to each other a heat shockpromoter nucleotide sequence, a synthetic intron nucleotide sequenceoptionally containing a Shine-Dalgamo sequence for efficient shuttlingbetween C. elegans and E. coli, optionally a nucleotide sequence codingfor a nuclear localisation signal or a secretion signal, e.g selectedfrom naturally occurring signal sequences, such as from C. elegans orthe signal sequence of the protein or nucleic acid molecule that is tobe expressed, a nucleotide sequence coding for a recognisable tag,optionally a nucleotide sequence coding for a fluorescent protein, anucleotide sequence coding for a protease cleavage site, a multiplecloning site containing a nucleotide sequence coding for an eukaryotic,such as human, protein or a nucleic acid molecule, and a nucleotidesequence coding for termination of translation.

The nucleotide sequence order in the plasmid may be modified so that themultiple cloning site is followed by the nucleotide sequence coding fora protease cleavage site, the optional nucleotide sequence coding for afluorescent protein, optionally the nucleotide sequence coding for anuclear localization signal or a secretion signal and the nucleotidesequence coding for a recognizable tag.

Examples of the nucleotide sequence coding for a protease cleavage siteinclude cleavage sites for the proteases TEV, Thrombin and Factor Xa.

In an embodiment of the plasmid vector, the synthetic intron nucleotidesequence contains the Shine-Dalgamo sequence AGGAG, the nucleotidesequence coding for a nuclear localization signal is SEQ ID NO: 3, thesequence coding for a recognizable tag is a sequence coding for a 6-Histag, a 10His tag or a 12-His tag, i.e. 6, 10 or 12 histidine residuesthat enable easy purification on Ni chelating columns, the nucleotidesequence coding for a fluorescent protein is a nucleotide sequencecoding for the green fluorescent protein with the sequence SEQ ID NO: 8,the nucleotide sequence coding for a protease cleavage site is asequence coding for a protease cleavage site, that enables latercleaving off of the 6, 10 or 12 histidine residues.

In a preferred embodiment the plasmid, lacking a nucleotide sequencecoding for an eukaryotic protein or a nucleic acid molecule, has thenucleotide sequence SEQ ID NO: 1. The plasmide has no nucleotidesequence coding for a nuclear localization signal. The artificial intronstarts at 480 and ends at 521, (gtatgtttcga atgatactaa cataacatagaacattttca g), then follows the 6-His-tag sequence, 547 to 564, (cat caccat cac cat cac), and a linker sequence, 565 to 594, connects theHis-tag to the sequence coding for the TEV protease recognition site:595 to 618. Then comes the multiple cloning site (MCS)(start at 619).

In another preferred embodiment the plasmid, lacking a nucleotidesequence coding for an eukaryotic protein or a nucleic acid molecule,has the nucleotide sequence SEQ ID NO: 2. The artificial intron startsat 480 and ends at 521 (gtatgtttcga atgatactaa cataacatag aacattttca g),then follows the nucleotide sequence coding for a nuclear localisationsignal(NLS): start at pos 533, end at 580 (ctagtgctca gaaaaaatgactgctccaaa aagaagcgt aaggtgcc). After the NLS comes the 6-His-tagsequence 588 to 605 (catcaccatc ccatcac). A linker sequence (606 to 635)connects it to the sequence coding for the TEV protease recognitionsite: 636 to 656, which is followed by the multiple cloning site (startat 658).

For cloning purposes, the NLS used in the plasmids is a bit longer thanthe essential NLS DNA sequence. (The NLS was cloned into the plasmidpre-cut with the restriction enzymes Nhel and Ncol). The essential NLSsequence (558 to 568: ccaaagaagaagcgtaaggtgcc c, [the last c comes fromthe Ncol cut vector]) translates into the protein sequence PKKKRKV, thatis recognized by the nuclear import machinery.

The nuclear localization signal, NLS, is useful for the followingreason. When the nematodes are heat shocked and in such a way forced toover-produce the desired human proteins, it may be safer to direct theproduced protein into the cell nucleus. This is exactly what the NLSdoes. The advantage of transporting proteins into the nucleus is thatthere are no proteases present. These proteases can be present in thecytoplasm and proteases are especially concentrated in organelles calledlysosomes. By using a NLS to send the expressed proteins into thenucleus, they are transported in one piece away from potentiallydangerous proteases.

The DNA sequence of the green fluorescent protein, GFP, containsadditionally three introns in the version in SEQ ID NO: 8. It isprimarily used as a luminescent marker to “visualize” that C. elegansexpresses the desired protein(s). In the plasmids where it is present,it follows the 6His-tag: e.g. 6His-GFP-TEV-MCS when incorporated intoplasmid SEQ ID NO: 1, or NLS-6His-GFP-TEV-MCS when incorporated intoplasmid SEQ ID NO: 2.

In yet another preferred embodiment the plasmid, lacking a nucleotidesequence coding for an eukaryotic protein or a nucleic acid molecule,has the nucleotide sequence SEQ ID NO: 9. The order of the sequences inthe plasmid is modified in relation to the sequences in the plasmid SEQID NO:1 and the green fluorescent protein with the sequence SEQ ID NO: 8is included. The sequences come in the order MCS-TEV-GFP-6His.

In still another preferred embodiment the plasmid, lacking a nucleotidesequence coding for an eukaryotic protein or a nucleic acid molecule,has the nucleotide sequence SEQ ID NO:10. The order of the sequences inthe plasmid is modified in relation to the sequences in the plasmid SEQID NO: 2 and the green fluorescent protein with the sequence SEQ ID NO:8 is included. The sequences come in the order MCS-TEV-GFP-NLS-6His.

Inserting the green fluorescent protein (GFP) after the multiple cloningsite (MCS) makes it possible to have a fast check for proper proteinfolding. If the expressed protein of interest, such as a human growthfactor, does not fold properly, it will not allow the green fluorescentprotein that follows after to fold properly either and as a consequenceone does not see green fluorescence. This is a fast test for proteinfolding. In addition, GFP can be used as a purification tag, either byusing ion exchange chromatography following established GFP protocols,or as an affinity tag using immobilized anti-GFP anti-bodies.

The preferred plasmids of the invention are intend to be used in E. colias well. Therefore they are designed as “shuttle vectors”. The onlymodification to the above disclosed plasmid sequences is that aso-called “Shine-Dalgarno” sequence is centered about 10 nucleotidesbefore the start codon of transcription, ATG. The Shine-Dalgarnosequence, AGGAG, is a translational initiation signal for E. coli anddoes not affect C. elegans.

Thus, in plasmid SEQ. ID NO: 1 the Shine-Dalgamo sequence is centered 10nucleotides before the ATG-6His; in plasmid SEQ ID NO: 2 theShine-Dalgamo sequence is centered 10 nucleotides before the ATG-NLS;and in plasmids SEQ ID NO: 9 and 10 the Shine-Dalgamo sequence iscentered 10 nucleotides before the MCS (the MCS contains an ATG).

The plasmids SEQ ID NO: 1, 2, 9 and 10 contain a protein that makes E.coli bacteria resistant to antibiotica, e.g. ampicillin or carbicillinor to kanamycin. However, these proteins have no effect on C. elegans(e.g. they do not provide any antibiotica resistance to C. elegans).

In a most preferred embodiment of the invention the nucleotide sequencecoding for a human protein is a sequence coding for a human growthfactor protein. Specific examples of some human growth factor proteinsare:

-   SEQ ID NO: 4, the sequence of a growth factor called Wnt2b (Homo    sapiens wingless-type MMTV integration site family, member 2B),-   SEQ ID NO: 5, the sequence of a growth factor called FGF10 (Homo    sapiens keratinocyte growth factor 2 (FGF10)),-   SEQ ID NO: 6, the sequence of a growth factor called KLS (Homo    sapiens KIT ligand soluble fraction), and-   SEQ ID NO: 7, the sequence of a growth factor called BMP10 (Homo    sapiens bone morphogenetic protein 10).

Another aspect of the invention is directed to a method of producingeukaryotic such as human, proteins or nucleic acid molecules innematodes comprising the steps of injecting one or several plasmidvectors, preferably simultaneously, according to the invention into thegonad of C. elegans hemaphrodites, cultivating the nematodes in a growthmedium at a temperature of below 25° C., followed by shifting the growthtemperature to values between 30 and 33° C. for induction of protein ornucleic acid molecule expression in several hundred somatic cells, withhighest expression levels in neuronal and epidermal cells, and isolatingthe eukaryotic proteins or nucleic acid molecules from said cells.

In an embodiment of the method of the invention the growth mediumcomprises bacteria, such as E. coli, as feed for the nematodes. Since C.elegans can feed exclusively on bacteria dispersed in minimal media,this fact can be exploited to label proteins produced in C. elegans. Byfeeding the worms bacteria that were previously labeled (e.g. with ²H,¹³C, ¹⁵N, Se-Met, Se-Cys or certain non-natural amino acids forexpression of proteins and ²H, ¹³C and ¹⁵N for expression of nucleicacid molecules) according to existing protocols, the respective labelwill be incorporated into newly produced proteins and nucleotides.

In a preferred embodiment the isolation is performed, in case theplasmid includes a nucleotide sequence coding for a nuclear localizationsignal, by carefully opening the cells of the nematodes, e.g. by using a“bead beater” (=a blender filled with small zirconium beads)—therebyleaving the cell nuclei intact which contain the expressed proteins ornucleic acid molecules. This is followed by separating the cell nucleiand by dissolving the nuclear membrane to release the expressed proteinsand subjecting the mixture to chromatographic purification. For proteinsthis includes a stationary phase specifically binding to therecognizable tag, e.g. 10His-tagged protein to Ni-chelating beads packedinto a purification column, followed by washing off unspecifically boundproteins, and elution under conditions releasing the eukaryotic, such ashuman, protein-from the column e.g. an imidazole gradient that releasesthe 10-His-tagged protein. The recognizable tag is then cleaved off bysupplying the specific protease corresponding to the protease cleavagesite encoded by the plasmid used and having an uncleavable recognizabletag, such as a 6-His-tag, and at the same time performing dialysisagainst a low concentration of the agent that releases the tag from thestationary phase, (suitably about 10-50 mM if the elution was done with400-700 mM imidazole), transferring the cut mixture containing theeukaryotic protein with the cut off tag and the protease that itself hasan uncleavable recognizable tag, e.g. 6-His-tag, onto a fresh tagspecific column, eluting the column to obtain an eluate containing theeukaryotic protein leaving the cut off recognizable tag, e.g. 10-Histag, and recognizably tagged, e.g. 6-His-tagged protease bound to thestationary phase.

In another preferred embodiment the isolation is performed, in case theplasmid lacks a nucleotide sequence coding for a nuclear localisationsignal, by mashing the nematodes, to release the expressed eukaryoticproteins and subjecting the mixture to chromatographic purification witha stationary phase specifically binding to the recognizable tag,followed by washing and elution under conditions releasing theeukaryotic protein from the stationary phase, as exemplified in thepreceding paragraph.

In case the plasmid used lacks a nuclear localization signal, or anextra precaution to prevent the eukaryotic protein from being attackedby unspecific proteases that can be present in the cell is desired, e.g.for cases where the proteins are degradation sensitive, an alternativeor complementary way of protecting the expressed proteins againstprotease degradation is to inject a separate plasmid, e.g. SEQ ID NO:1or 2 lacking the nucleotide sequence coding for a protease cleavagesite, and containing a nucleotide sequence coding for a general proteaseinhibitor, such as alpha2-Macroglobulin (α2-M), SEQ ID NO: 11.

Thus, in an additionally preferred embodiment of the method of theinvention, the method comprises additionally injecting a plasmid vectorcomprising operably linked to each other a heat shock promoternucleotide sequence, a synthetic intron nucleotide sequence optionallycontaining a Shine-Dalgamo sequence, optionally a nucleotide sequencecoding for a nuclear localisation signal, a nucleotide sequence codingfor a recognisable tag, optionally a nucleotide sequence coding for afluorescent protein, a nucleotide sequence coding for a general proteaseinhibitor, such as the general protease inhibitor SEQ ID NO:11 codingfor α2-Macroglobulin, and a nucleotide sequence coding for terminationof translation, for co-expression of the general protease inhibitor. Inthis case, the recognisable tag remains on the expressed generalprotease inhibitor and it will be bound to the tag specific column(together with the (e.g. 6-His-) tagged protease and the cleaved off(e.g. 10-His-) tag from the eukaryotic protein in the second and lastspecific column step).

The C. elegans expression system, including the plasmids and the methodof producing eukaryotic, such as human, proteins or nucleic acidmolecules in nematodes of the invention, is particularly suitable forlarge scale production of ultra-pure recombinant eukaryotic proteins, inparticular human growth factors. Proteins produced will contain themodifications that are typical for higher organisms (eukaryotes), suchas acetylation, N- and O-linked glycosylation and, acylation,phosphorylation and cleavage of signal sequences etc. Thesemodifications are crucial for the specificity of a medically interestingprotein in signalling pathways. Examples of human (eukaryotic) proteinsthat may be produced on an industrial scale with the present inventioninclude: human growth factors, growth factor receptors (membrane boundor soluble part) for basic research on stem cells and for medicalapplications. In addition monoclonal antibodies, G-proteins, G-proteincoupled receptors. In particular, the system is designed to allow forshuttling between C. elegans and E. coli in order to study the effectsof post-translational modifications. It also allows the labelling of C.elegans produced proteins and nucleic acid molecules with identifiablemarkers (e.g. ²H, ¹³C, ¹⁵N, Se-Met, Se-Cys, etc.) for NMR and X-raycrystallographic studies, by feeding the nematodes with pre-labelledbacteria. Further, the expression of eukaryotic proteins, such as human,proteins or nucleic acid molecules in C. elegans can be directed to theprotective protease reduced environment of the cell nucleus or tocertain compartments of the cell, depending on the chosen signalpeptide. The system also allows simultaneous expression of proteins frommore than 20 plasmids if the reconstruction of large, multi-subunitprotein-RNA complexes such as polymerases, telomerase and splicingfactor complexes is required.

The invention will now be further illustrated by description ofexperiments, and it should be understood that the scope of the claims isnot restricted to any specifically mentioned details.

Experiments

All manipulations of C. elegans worms were performed using techniquesdescribed in Methods in Cell Biology, vol 84; Caenorhabditis elegans:modern biological analysis of an organism, ed. Epstein and Shakes,Academic Press, 1995, or using minor modifications of the methodsdescribed therein.

Transgenic C. elegans strains were constructed by injection of plasmidDNA into worms of the genotype unc-36(e251);hmp-1-(zu278)/daf-11(m8ts)sma-1(e30) together with plasmids encodingunc-36(+) and hmp-1(+) (Costa et al., Journal of Cell Biology 141:297-308 1998). hmp-1(zu278) causes an embryonic lethal phenotype (Costaet al. 1998). Lines were established of the genotypes unc-36(e251);hmp-1(zu278); svEx[unc-36(+) hmp-1(+) hs-gen-1(+)] where ‘gen-1’ denotesthe gene encoding the human growth factor.

Worms were heat shocked at 33° C. for two hours by using establishedprocedures. (Stringham E G and Candido, E P M Environmental Toxicologyand Chemistry 13: 1211-1220 1994).

Expression Vector Construct

In order to express proteins in C. elegans we have built four plasmidvectors based on the plasmide pPD49,78-umu, SEQ ID NO: 1,2,9 and 10,that allows the heat inducible expression of genes in many differentcells. The vector contains a C. elegans heat shock promoter for theectopic expression of foreign proteins. The promoter is inactive below25° C. However, shifting the growth temperature to values between 30 and33° C. results in induction of protein expression at high level inseveral hundred somatic cells with highest expression levels in neuronaland epidermal cells.

The vectors are designed to make translational fusions. Downstream ofthe ATG is a sequence encoding a 6-His-tag followed by a TEV proteasecleavage site and a multiple cloning site in plasmids SEQ ID NO: 1 and2.

Micro-Injection and Selection

In order to determine the expression level of single protein, wegenerated several plasmids each containing a different test genetogether with appropriate genetic selection markers on separateplasmids.

We injected plasmids into hermaphrodites of the genotype unc-36(e251);hmp-1 (zu278)/daf-11 (m8ts)sma-1(e30) together with plasmids encodingunc-36(+) and hmp-1(+). hmp-1(zu278) causes an embryonic lethalphenotype (Costa et al. 1998). Lines were established of the genotypesunc-36(e251); hmp-1(zu278); svEx[unc-36(+) hmp-1(+) hs-gen-1(+)] where‘gen-1’ denotes the gene to be tested.

Recombination between different plasmids of more then 600 bp occurs invivo and transformed first generation F1 progeny are obtained in whichthe different injected plasmids together form an episome (referred to asan extrachromosomal array). Approximately 1 in 10 F1 transformants giverise to stable lines in which the extrachromosomal array is transmittedfrom generation to generation without change. Extrachromosomal arraysare relatively stable mitotically so that in any one transformed animalmost cells contain the array. By using appropriate co-injection markersencoding essential genes it is possible to obtain strains in which alladult and larval worms in the population contain the array.

Protein Expression

C. elegans is grown from liquid medium in Erlenmeyer flasks on atemperature controlled shaker at 20° C. The liquid growth mediumcontains the slow growing E. coli strain OP50 on which the nematodesfeed. The worms are grown during 7 days after which shifting the growthtemperature from 20° C. to 33° C. induces protein production under thecontrol of a heat shock promoter.

The yield of wet worms grown from one liter of initial culture is about10 g. The green fluorescence protein (GFP) is expressed in about 25% ofthe cells and leads to a visible florescence signal that is seen underthe fluorescence light microscope.

The yield of pure protein from a starting material of 10 to 20 gcultured and induced worms is roughly 0.2 to 1.0 mg.

The human growth factor called Wnt2b (Homo sapiens wingless-type MMTVintegration site family, member 2B), was successfully expressed in theworms from the plasmide SEQ ID NO: 1 comprising in the multiple cloningsite the nucleotide sequence SEQ ID NO: 4.

In short, the nematodes are grown from simple liquid medium. They growin 25 fermenters or in Erlenmeyer flasks. The worms have a generationtime of about 2-3 days and adult worms grow to about 1 mm in length.Each hermaphrodite produces about 300-500 eggs per generation. They canbe repeatedly stored for long periods of time at −80° C. Stable linescan be selected after injection of up to twenty different plasmids intothe gonad of hemaphrodites.

1. Plasmid vector for expression in Caenorhabditis elegans and in Escherichia coli a comprising in the 5′ to 3′ direction of transcription operably linked to each other a heat shock promoter nucleotide sequence, a synthetic intron nucleotide sequence containing a Shine-Dalgarno sequence, optionally a nucleotide sequence coding for a nuclear localisation signal or a secretion signal, a nucleotide sequence coding for a recognisable tag, optionally a nucleotide sequence coding for a fluorescent protein, a nucleotide sequence coding for a protease cleavage site, a multiple cloning site containing a nucleotide sequence coding for an eukaryotic, such as human, protein or a nucleic acid molecule, and a nucleotide sequence coding for termination of translation.
 2. Plasmid vector according to claim 1, wherein the a nucleotide sequence order is modified so that the multiple cloning site is followed by the nucleotide sequence coding for a protease cleavage site, the optional nucleotide sequence coding for a fluorescent protein, optionally the nucleotide sequence coding for a nuclear localisation signal or a secretion signal and the nucleotide sequence coding for a recognisable tag.
 3. Plasmid vector according to claim 1, wherein the synthetic intron nucleotide sequence contains the Shine-Dalgarno sequence AGGAG, the nucleotide sequence coding for a nuclear localisation signal is SEQ ID NO: 3, the sequence coding for a recognisable tag is a sequence coding for a 6-His, 10-His or 12-His tag, the nucleotide sequence coding for a fluorescent protein is a nucleotide sequence coding for the green fluorescent protein with the sequence SEQ ID NO: 8, the nucleotide sequence coding for a protease cleavage site is a sequence coding for a TEV protease cleavage site.
 4. Plasmid vector according to claim 1, wherein the plasmid, lacking a nucleotide sequence coding for an eukaryotic protein or a nucleic acid molecule, has the nucleotide sequence SEQ ID NO:
 1. 5. Plasmid vector according to claim 1, wherein the plasmid, lacking a nucleotide sequence coding for an eukaryotic protein or a nucleic acid molecule, has the nucleotide sequence SEQ ID NO:
 2. 6. Plasmid vector according to claim 2, wherein the plasmid, lacking a nucleotide sequence coding for an eukaryotic protein or a nucleic acid molecule, has the nucleotide sequence SEQ ID NO:
 9. 7. Plasmid vector according to claim 2, wherein the plasmid, lacking a nucleotide sequence coding for an eukaryotic protein or a nucleic acid molecule, has the nucleotide sequence SEQ ID NO:
 10. 8. Plasmid vector according to claim 1, wherein the nucleotide sequence coding for an eukaryotic protein is a sequence coding for a human growth factor protein.
 9. Plasmid vector according to claim 8, wherein the sequence coding for a human growth factor protein is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO:
 7. 10. Method of producing eukaryotic, such as human, proteins or nucleic acid molecules in nematodes comprising the steps of injecting one or several plasmid vectors according to claim 1 into the gonad of C. elegans hermaphrodites, cultivating the nematodes in a growth medium at a temperature of below 25° C., followed by shifting the growth temperature to values between 30 and 33° C. for induction of protein or nucleic acid molecule expression in several hundred somatic cells, with highest expression levels in neuronal and epidermal cells, and isolating the eukaryotic proteins or nucleic acid molecules from said cells.
 11. Method according to claim 10, wherein the growth medium comprises optionally labelled bacteria as feed for the nematodes.
 12. Method according to claim 11, wherein the bacteria are labelled with identifiable markers selected from the group consisting of ²H, 13C, ¹⁵N, Se-Met, Se-Cys and non-natural amino acids for expression of proteins and selected from ²H, ¹³C and ¹⁵N for expression of nucleic acid molecules.
 13. Method according to claim 10, wherein the isolation is performed, in case the plasmid includes a nucleotide sequence coding for a nuclear localisation signal, by carefully opening the nematodes so that the cells containing the expressed proteins are opened, leaving the cell nuclei intact, followed by isolating the cell nuclei and dissolving the nuclear membrane to release the expressed proteins or nucleic acid molecules and subjecting the mixture to chromatographic purification including, for proteins, a stationary phase specifically binding to the recognizable tag, followed by washing, and elution under conditions releasing the eukaryotic protein from the recognizable tag protein by supplying a specific protease corresponding to the protease cleavage site encoded by the plasmid used and having an uncleavable recognizable tag, and at the same time performing dialysis against a low concentration of an agent that releases the tag from the stationary phase, transferring the cut mixture containing the human protein with the cut off tag and the protease with the uncleavable recognizable tag onto a fresh tag-specific column, eluting the column to obtain an eluate containing the eukaryotic protein, leaving the cut off recognizable tag, and recognizable tag-containing protease bound to the stationary phase.
 14. Method according to claim 10, wherein the isolation is performed, in case, the plasmid lacks a nucleotide sequence coding for a nuclear localisation signal, by mashing the nematodes, to release the expressed proteins or nucleic acid molecules and subjecting the mixture to chromatographic purification including, for proteins, a stationary phase specifically binding to the recognizable tag, followed by washing and elution under conditions releasing the eukaryotic protein from the stationary phase.
 15. Method according to claim 10, comprising additionally injecting a plasmid vector comprising operably linked to each other a heat shock promoter nucleotide sequence, a synthetic intron nucleotide sequence optionally containing a Shine-Dalgarno sequence, optionally a nucleotide sequence coding for a nuclear localisation signal, a nucleotide sequence coding for a recognisable tag, optionally a nucleotide sequence coding for a fluorescent protein, a nucleotide sequence coding for a general protease inhibitor, and a nucleotide sequence coding for termination of translation, for co-expression of the general protease inhibitor.
 16. Method according to claim 15, wherein the nucleotide sequence coding for a general protease, inhibitor is SEQ ID NO :11 coding for a2-Macroglobulin.
 17. Plasmid vector according to claim 3, wherein the plasmid, lacking a nucleotide sequence coding for an eukaryotic protein or a nucleic acid molecule, has the nucleotide sequence SEQ ID NO:
 1. 18. Plasmid vector according to claim 3, wherein the plasmid, lacking a nucleotide sequence coding for an eukaryotic protein or a nucleic acid molecule, has the nucleotide sequence SEQ ID NO:
 2. 19. Plasmid vector according to claim 3, wherein the plasmid, lacking a nucleotide sequence coding for an eukaryotic protein or a nucleic acid molecule, has the nucleotide sequence SEQ ID NO:
 9. 20. Plasmid vector according to claim 3, wherein the plasmid, lacking a nucleotide sequence coding for an eukaryotic protein or a nucleic acid molecule, has the nucleotide sequence SEQ ID NO:
 10. 